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Research Article
RUNX3, A Novel Tumor Suppressor, Is Frequently Inactivated in
Gastric Cancer by Protein Mislocalization
1,2 2 2,3 1 2 1
Kosei Ito, Qiang Liu, Manuel Salto-Tellez, Takashi Yano, Kotaro Tada, Hiroshi Ida,
2 2 1,2 4 2
Canhua Huang, Nilesh Shah, Masafumi Inoue, Andrea Rajnakova, Kum Chew Hiong,
2 2 2 3 4 1,2
Bee Keow Peh, Hwan Chour Han, Tomoko Ito, Ming Teh, Khay Guan Yeoh, and Yoshiaki Ito
1
Institute of Molecular and Cell Biology, Proteos; 2Oncology Research Institute; and Departments of 3Pathology and 4Medicine, Faculty of
Medicine, National University of Singapore, Singapore
Abstract diffuse-type gastric cancers. The oncogenic activation of b-catenin
Loss of RUNX3 expression is suggested to be causally related (17-27% in differentiated type) and K-ras (0-18% in both diffuse and
to gastric cancer as 45% to 60% of gastric cancers do not differentiated types) have been found in human gastric cancer. In
express RUNX3 mainly due to hypermethylation of the RUNX3 addition, the c-erbB2 or c-met gene is amplified in f10% of both
promoter. Here, we examined for other defects in the cancer types. Among tumor suppressor genes, APC mutations are
properties of RUNX3 in gastric cancers that express RUNX3. found frequently in gastric adenomas but only rarely in gastric
Ninety-seven gastric cancer tumor specimens and 21 gastric cancers. Similarly, mutation of the p16 gene is infrequent (3).
cancer cell lines were examined by immunohistochemistry Mutations in p53 have been reported for gastric cancers of the
using novel anti-RUNX3 monoclonal antibodies. In normal diffuse and intestinal types, but the role of p53, if any, in gastric
gastric mucosa, RUNX3 was expressed most strongly in the carcinogenesis remains obscure.
nuclei of chief cells as well as in surface epithelial cells. In We reported recently that loss of expression of RUNX3 is causally
chief cells, a significant portion of the protein was also found related to the genesis and progression of gastric cancer. About 45%
in the cytoplasm. RUNX3 was not detectable in 43 of 97 (44%) to 60% of surgically resected gastric cancer specimens and cell lines
cases of gastric cancers tested and a further 38% showed derived from these cancers do not express RUNX3 due to
exclusive cytoplasmic localization, whereas only 18% showed hemizygous deletion of the gene or hypermethylation of its
nuclear localization. Evidence is presented suggesting that promoter region. Inactivation of RUNX3 appears to occur at an
transforming growth factor-B is an inducer of nuclear early stage as well as during progression, because silencing of
translocation of RUNX3, and RUNX3 in the cytoplasm of RUNX3 has been observed in 40% of stage I and 90% of stage IV
cancer cells is inactive as a tumor suppressor. RUNX3 was gastric cancers. A mutation found in a gastric cancer patient,
found to be inactive in 82% of gastric cancers through either RUNX3 (R122C), which causes a single amino acid substitution
gene silencing or protein mislocalization to the cytoplasm. In within the conserved DNA-binding domain, completely abolishes
addition to the deregulation of mechanisms controlling gene the tumor suppressor activity of RUNX3 in a nude mouse assay.
expression, there would also seem to be at least one other Hyperplasia of the gastric epithelium, as observed in a Runx3À/À
mechanism controlling nuclear translocation of RUNX3 that experimental mouse system, seems to be caused by decreased
is impaired frequently in gastric cancer. (Cancer Res 2005; sensitivity to transforming growth factor-h (TGF-h), which inhibits
65(17): 7743-50) cell cycle progression and induces apoptosis. Furthermore, experi-
ments with stomach epithelial cell lines isolated from Runx3+/+ and
Introduction Runx3À/À mice with the p53À/À background revealed that only
those lines derived from Runx3À/Àp53À/À mice were tumorigenic
Gastric cancer is the second leading cause of cancer death
in nude mice (4). Although these results strongly suggested that
worldwide, with 560,000 new cases and 405,000 deaths each year.
RUNX3 is a gastric cancer tumor suppressor and that its loss is
The prognosis for stage IV gastric cancer is still poor, with a 5-
involved in roughly half of the cases of gastric cancer, we assumed
year survival rate of f10% (1). However, with advances in
that it functions normally in the remaining cases. We report in this
diagnostic techniques and treatment methods, the outlook for
gastric cancer has considerably improved. If it is diagnosed at an article that this assumption was likely incorrect.
early stage, the prognosis is favorable and the disease may even Hanai et al. (5) have shown that RUNX3 forms complexes with
be curable. receptor-regulated Smads (R-Smads) that regulate target gene
Despite its prominence, the molecular mechanisms leading to expression; RUNX3 is thus a downstream target of the TGF-h
gastric carcinogenesis are still poorly understood and only a few signaling pathway. This signaling pathway is often called a tumor
genes have been clearly implicated. Germ line mutations of E- suppressor pathway, because certain of its components are
cadherin were first found in a large family from New Zealand in frequently genetically or epigenetically altered in many types of
which diffuse-type gastric cancers developed at an early age (2). cancers, especially those of the gastrointestinal tract (6). TGF-h is
Subsequently, the somatic mutations were also observed in sporadic a multifunctional growth factor that has profound regulatory
effects on many developmental and physiologic processes. In TGF-
b1-null animals, gastric epithelial proliferation is stimulated and
Requests for reprints: Yoshiaki Ito, Institute of Molecular and Cell Biology, epithelial hyperplasia is observed together with dysregulation of
Proteos, 61 Biopolis Drive, Singapore 138673. Phone: 65-6586-9646; Fax: 65-6779-1117; differentiation and intestinal metaplasia (7). These phenotypes are
E-mail: itoy@imcb.a-star.edu.sg.
I2005 American Association for Cancer Research. also seen in Runx3À/À gastric epithelial cells (8). Thus, TGF-h
doi:10.1158/0008-5472.CAN-05-0743 signaling may regulate the growth and differentiation of gastric
www.aacrjournals.org 7743 Cancer Res 2005; 65: (17). September 1, 2005
Cancer Research
epithelial cells at least partly by mediating molecular interactions lots of FCS contain TGF-h, which affects subcellular localization of RUNX3
between RUNX3 and R-Smads (8, 9). RUNX3 is located in the 1p36 in SNU16 cells). The gastric cancer cell lines IM95 and Hs746T were
locus, a region that is considered to carry a tumor suppressor maintained in DMEM supplemented with 10% fetal bovine serum.
SNU16 cells were transfected with pcDNA3.1/HisC, pcDNA-Flag-RUNX3
gene(s) implicated in various types of cancers, especially those of
(1-187 amino acids; ref. 17), and pEF-Bos-neo-RUNX3-AS using Lipofect-
the gastrointestinal tract. Therefore, at least one of the long amine 2000 and Lipofectamine Plus reagents (Invitrogen, Carlsbad, CA) to
sought-after tumor suppressors on 1p36 could be RUNX3, which generate stable transfectants, control SNU16 cells, SNU16 cells expressing
seems to be an integral component of the TGF-h tumor suppressor RUNX3 (1-187 amino acids), and SNU16 cells expressing antisense RUNX3
pathway (10). DNA, respectively. pEF-Bos-neo-RUNX3-AS was constructed by subcloning a
In this study, monoclonal antibodies were generated for the RUNX3 cDNA fragment (accession no. Z35278; ref. 14) into the XbaI site of
immunohistochemical analysis of RUNX3 in human gastric tissue pEF-Bos-neo (18) in reverse orientation by blunt end ligation. Stable
sections. Surprisingly, in 67% of 55 human gastric cancer speci- transfectants were selected on 0.5 mg/mL G418 (Invitrogen). Independent
mens and in all 5 gastric cancer cell lines expressing RUNX3 thus clones that stably expressed Flag-RUNX3 (1-187 amino acids) and reduced
levels of RUNX3 mRNA were identified by Western blot analysis using an
far tested, RUNX3 was found in the cytoplasm. Because RUNX3 is a
anti-Flag monoclonal antibody (M2) and by reverse transcription-PCR (RT-
transcription factor, these results suggest that it is mislocalized
PCR) using the primers 5V -CTACGGGACATCCTCTGGCTCC-3V (1,008-1,029)
and thus nonfunctional in a large fraction of gastric cancer cases. and 5V -CATCTCTGCCAGCAGCGTGCTG-3V (1,825-1,846) to amplify 839 bp
Therefore, RUNX3 seems to play a more prominent role in gastric of RUNX3 cDNA, which is not included in pEF-Bos-neo-RUNX3-AS,
carcinogenesis than previously estimated. respectively.
Immunohistochemistry, immunocytochemistry, immunoblotting,
and in situ hybridization. Ten percent formalin-fixed human gastric
Materials and Methods tissues or 4% paraformaldehyde-fixed tumors formed by 1 Â 107 SNU16
Generation of anti-RUNX3 monoclonal antibodies. Polypeptide or MKN74 cells in nude mice 60 days after inoculation were embedded
antigens were expressed in an Escherichia coli system and purified with in paraffin and cut into 5 Am sections. Alternatively, 5 Am sections of
the QIAexpressionist protein purification kit (Qiagen, Germany). Purified tissue microarrays from gastric cancer and normal counterparts,
antigens were suspended as 25 Ag polypeptide in 0.1 mL PBS, which was constructed as described elsewhere (19), were prepared. The rehydrated
emulsified with 0.1 mL Freund’s complete adjuvant and injected s.c. into sections were warmed in a target retrieval solution (DAKO, Denmark) at
7-week-old female BALB/c mice. Booster injections of 25 and 12 Ag 96jC for 40 minutes. Rehydrated tissue sections or 4% paraformaldehyde-
polypeptide in 0.1 mL PBS with 0.1 mL incomplete adjuvant were given fixed cells on the slides were treated with a serum-free blocking solution
i.p. or s.c. at days 14 and 60, respectively. On day 69, animals were bled (DAKO) and incubated at 4jC overnight with 1 Ag/mL R3-6E9 in the
and the presence of antibodies against RUNX3 was determined by Western absence or presence of 0.5 Ag/mL purified antigens (amino acids 8-72 or
blot analysis (see below). On day 70, mice were boosted again with an i.v. 191-300) in a diluent solution (DAKO). A peroxidase-3,3V -diaminobenzi-
injection of 12 Ag polypeptide in 0.2 mL PBS. On day 74, spleens were dine–based detection system (EnVision+ kit, DAKO) was used to detect
removed from immunized mice and the splenocytes (lymphocytes) were the immunoreactivity of R3-6E9 on the sections. Biotinylated anti-mouse
fused with the SP2-K13 murine myeloma cell line, a subclonal line derived IgG (Vector, Burlingame, CA) and fluorescein-avidin D (Vector) were used
from SP2/0-Ag14 myeloma cells, as described (11), using 50% polyethylene for immunofluorescence imaging. Cases with the majority of the cells
glycol (4000). (>80%) showing RUNX3 expression (nuclear or cytoplasmic) were
Individual hybridomas were cloned by the limiting dilution technique counted positive. Cases with no expression or only with minimal and
with thymocyte feeder cells from BALB/c mice. Hybridoma culture fluids equivocal expression in a minority of cells (<10%) were counted negative.
were screened for secreted antibodies against RUNX3 by Western blot Whole cell extract was prepared by sonicating cells in the presence of
analysis using extracts from COS7 cells exogenously expressing human full- 9 mol/L urea and 2% Triton X-100. Nuclear and cytoplasmic extracts were
length RUNX3. Three cycles of cloning and recloning to screen hybridoma prepared using NE-PER Nuclear and Cytoplasmic Extraction reagents
cells were done to obtain cells that secrete anti-RUNX3 antibodies and that (Pierce, Rockford, IL) and a 27-gauge needle. Twenty micrograms each of
show no reactivities to human RUNX1 and RUNX2. Subsequently, cells whole cell extracts or nuclear and cytoplasmic extracts from 5 Â 104 of
producing specific antibodies were adapted to serum-free culture and IgG SNU5 and RF48 cells were separated by 10% SDS-PAGE and subjected to
was purified by protein G-Sepharose. Western blot analysis with 0.05 Ag/mL R3-5G4.
Characterization of anti-RUNX3 monoclonal antibodies. Extracts of RUNX3 mRNA in paraffin sections of human gastric tissues was detected
COS7 cells harboring derivatives of the expression vector pEF-Bos that by in situ hybridization as described previously (4).
express human full-length RUNX1, RUNX2, or RUNX3 or murine full- Luciferase assay. SNU16 cells were transfected with (ThRE-WT)3,
length Runx1, Runx2, or Runx3 (refs. 12–16; the murine Runx3 cDNA, (ThRE-mP)3, and (ThRE-mS)3 luciferase reporter constructs, which contain
accession no. AF155880, was inserted into the EcoRI sites of pEF-Bos) three copies each of the wild-type TGF-h response element in the Ig Ca
were used as test antigens for examining antibody specificities. The promoter or of derivatives with mutations affecting the RUNX or Smad
extracts were separated by 10% SDS-PAGE and subjected to Western blot binding sites, respectively (5). Human TGF-h1 (2 Ag/mL; R&D Systems,
analysis. Minneapolis, MN) was added to the culture medium 48 hours after the
Extracts of COS7 cells harboring derivatives of the expression vector transfection. Luciferase activity was measured using the Dual-Luciferase
pcDNA3 that express Flag-tagged full-length RUNX3 or Flag-tagged Reporter Assay System (Promega, Madison, WI) and normalized to the
truncated forms of RUNX3 (1-187, 1-234, 1-283, 1-325, and 1-373 amino luciferase activity expressed by the pRL-TK vector.
acids; refs. 5, 17) were used as antigens for epitope mapping. The extracts
were separated by 10% SDS-PAGE and subjected to Western blot analysis.
Flag-tagged full-length RUNX3 or Flag-tagged truncated forms of RUNX3 Results
were visualized with an anti-Flag monoclonal antibody (M2; Sigma,
Isolation of monoclonal antibodies against RUNX3. To
St. Louis, MO).
Cell culture and stable transfection of SNU16 cells. The gastric cancer
generate anti-RUNX3 mouse monoclonal antibodies, 6Â His-
cell lines MKN1, MKN7, MKN28, MKN45, MKN74, AGS, NUGC3, SCH, tagged purified polypeptides consisting of 65 and 110 amino acids
KATOIII, Ist1, SNU1, SNU5, SNU16, GMK, TMK1, AZ521, NCI-N87, and from the human RUNX3 protein (amino acids 8-72 and 191-300,
Takigawa and a cell line derived from a B-cell lymphoma, RF48, were respectively) were used as antigens to immunize mice (see
maintained in RPMI 1640 supplemented with 10% fetal bovine serum (some Materials and Methods). Of >200 hybridoma clones producing
Cancer Res 2005; 65: (17). September 1, 2005 7744 www.aacrjournals.org
Cytoplasmic Retention of RUNX3
Figure 1. Immunodetection of RUNX3 in normal human gastric epithelial cells with the specific monoclonal antibody R3-6E9. A,left,top, Western blot analysis with
Rp-3D9 of human RUNX1 (lane 1), human RUNX2 (lane 2), human RUNX3 (lane 3 ), murine Runx1 (lane 4 ), murine Runx2 (lane 5 ), and murine Runx3 (lane 6 )
expressed in COS7 cells; left, bottom, Western blot analysis with R3-6E9; right,top, mapping the epitope recognized by R3-6E9 on RUNX3. Immunodetection of
Flag-tagged full-length (lane 1) and truncated forms of RUNX3 (lane 2, 1-373 amino acids; lane 3, 1-325 amino acids; lane 4, 1-283 amino acids; lane 5, 1-234 amino
acids; lane 6, 1-187 amino acids) by Western blot analysis with an anti-Flag monoclonal antibody; right,bottom, immunodetection of Flag-tagged full-length and
truncated forms of RUNX3 by Western blot analysis with R3-6E9. B, immunodetection of RUNX3 in normal human gastric epithelial cells in corpus and pyloric antra
with R3-6E9. Top and bottom boxed regions are enlarged on the right . Immunostaining with R3-6E9 without counterstaining in a section similar to the lower
enlarged region is shown (without hematoxylin). Arrows, parietal cells with weaker immunoreactivity than adjacent chief cells. C,left, immunodetection of RUNX3 in
normal human gastric epithelial cells in corpus and pyloric antra with R3-2A4 or normal mouse IgG. Counterstaining was done with hematoxylin; right, detection
of RUNX3 mRNA by in situ hybridization with a RUNX3 antisense or sense probe. D, immunostaining of normal gastric mucosa with R3-6E9 preincubated with purified
polypeptides, amino acids 8 to 72 or 191 to 300 of RUNX3. Bars, 200 Am.
specific antibodies against RUNX3 as detected by Western analysis, this epitope likely resides between amino acids 191 and 234. The
antibodies from two clones, R3-2A4 (raised against the amino acids specificity of R3-6E9 for RUNX3 was rigorously examined by several
8-72 antigen) and R3-6E9 (raised against the amino acids 191-300 assays (data not shown).
antigen), were found useful for immunohistochemical analysis. The Immunodetection of RUNX3 in normal human stomach
R3-5G4 clone, which reacts with the amino acids 191 to 300 antigen, epithelial cells with R3-6E9. We first determined which epithelial
was suitable for Western blot analysis. A previously isolated clone cell types in the adult human stomach express RUNX3. As shown in
raised against the conserved Runt domain of RUNX1, Rp-3D9, Fig. 1B, almost all stomach epithelial cells in both corpus and
recognizes the Runt domain of all three RUNX proteins equally pyloric antra were immunostained with R3-6E9. In particular, chief
well.5 cells and surface epithelial cells were stained most strongly, whereas
The specificity of the R3-6E9 antibody against RUNX3 proteins is parietal cells showed a lower level of expression (Fig. 1B). This
shown in Fig. 1A. R3-6E9 reacted only with human RUNX3 and expression pattern is consistent with that of RUNX3 mRNA as
mouse Runx3 but recognized the human protein more efficiently revealed by in situ hybridization using an antisense RUNX3 RNA
than the mouse protein (Fig. 1A). R3-6E9 reacted with all COOH- probe and a control sense probe (Fig. 1C), strongly supporting the
terminally truncated forms of RUNX3, except for RUNX3 (1-187 authenticity of the RUNX3 distribution revealed by R3-6E9. The
amino acids), indicating that the epitope recognized by R3-6E9 lies results of immunostaining with R3-2A4 were virtually identical to
within the 188– to 234–amino acid region (Fig. 1A). Because the those obtained with R3-6E9 (Fig. 1C). The immunoreactivity of R3-
antigen used to immunize mice was RUNX3 (191-300 amino acids), 6E9 on gastric mucosa was removed when R3-6E9 was preincubated
with the RUNX3 peptide (amino acids 191-300) used to raise this
antibody but not with amino acids 8 to 72 antigen for R3-2A4,
5
K. Ito, unpublished data. showing the specificity of R3-6E9 to its epitope (Fig. 1D).
www.aacrjournals.org 7745 Cancer Res 2005; 65: (17). September 1, 2005
Cancer Research
Interestingly, R3-6E9 detected a significant amount of RUNX3
protein in the cytoplasm as well as in the nuclei of chief cells
(pyloric gland cells in the pyloric antrum; Fig. 1B). RUNX3 was
localized primarily in the nuclei of surface epithelial cells (Fig. 1B).
The presence of a substantial amount of RUNX3 in the cytoplasm
suggests at least two possibilities: RUNX3 has an as yet unknown
function in the cytoplasm and/or it is retained in the cytoplasm in
an inert form until it is mobilized to the nucleus under appro-
priate conditions.
Cytoplasmic retention of RUNX3 in gastric cancer cells.
Paraffin sections of 97 clinical samples from gastric cancer patients
were subjected to immunohistochemistry with R3-6E9. We found
three types of staining pattern for RUNX3: (a) negative in the both
nucleus and cytoplasm (negative), (b) positive in the nucleus and
positive or negative in the cytoplasm (positive in nucleus), and (c)
positive in the cytoplasm and negative in the nucleus (positive in
cytoplasm). These three patterns were observed in both differen-
tiated type (intestinal type; Fig. 2A and C) and diffuse type (Fig. 2B)
of gastric cancer, and their frequencies are summarized in Table 1.
The fraction of gastric cancer cases in which RUNX3 was not
expressed was 44% (43 of 97 cases tested); this frequency is
consistent with our previous results obtained by in situ hybridiza-
tion (4). Surprisingly, 69% of RUNX3-positive gastric cancers (38% of
total cases) fell into the third class, as shown in Fig. 2 A (iii), B (iii),
and C, and only 18% fell into the second class.
Next, we examined the significance of the cytoplasmic expression
of RUNX3 in gastric cancers in more detail using gastric cancer–
derived cell lines. Western analysis indicated that the lines MKN1,
MKN45, SNU5, SNU16, RF48, and NCI-N87 expressed RUNX3,
whereas MKN28 and SNU1 did not (Fig. 3A). The subcellular
localization of RUNX3 in these lines was determined by immuno-
cytochemistry and cell fractionation. All RUNX3-positive cell lines,
except for RF48, showed a cytoplasmic localization of RUNX3
(Fig. 3B and C). The RUNX3-negative cell lines MKN28 and SNU1
did not immunoreact with R3-6E9 (Fig. 3C), suggesting that it
does not cross-react with any other protein in gastric cancer cells
at a significant level. The RF48 line, in which RUNX3 localizes to
the nucleus (Fig. 3B and C), was recently found to be derived from
a B-cell lymphoma rather than from a gastric cancer (20). Further- Figure 2. Immunodetection of RUNX3 in gastric cancer cells with R3-6E9.
more, RUNX3 exogenously expressed in NIH3T3 cells was found to A and C, sections prepared from differentiated (intestinal) gastric cancers.
localize to the nucleus (data not shown) in the same way that B, sections prepared from diffuse gastric cancers. Three types of staining
patterns for RUNX3 were observed: (i ) negative [A (i) and B (i)], (ii ) positive
exogenously expressed Runx1 and Runx2 localize to the nuclei of in nucleus [A (ii ) and B (ii )], and (iii ) positive in cytoplasm [A (iii ), B (iii ), and C ].
these cells as reported previously (21). These results show that the The boxed regions on top are enlarged on the bottom (A and B ). The boxed
cytoplasmic localization of RUNX3 is frequently and specifically region is enlarged on the right (C ). Counterstaining was done with hematoxylin.
Immunostaining without hematoxylin was done on a serial section (C ). Bars,
observed in gastric cancer cells. 200 Am.
Nuclear translocation of RUNX3 on stimulation by trans-
forming growth factor-B. RUNX3 is a downstream target of the
TGF-h signaling pathway and most gastric cancer–derived cell antisense DNA against RUNX3 (Fig. 4B). RUNX3 (1-187) is a
lines are resistant to stimulation by TGF-h, suggesting that this dominant-negative form of RUNX3, which has an intact DNA-
pathway is frequently impaired in gastric cancers. The gastric binding domain (the Runt domain) but lacks the transactivation
cancer cell line SNU16, which expresses Smad2, Smad3, Smad4, domain (5, 23). Furthermore, RUNX3 (1-187) does not have tumor-
and a wild-type TGF-h type II receptor (22), is exceptional among suppressive effects on gastric cancer cells (17). In the presence of
such cell lines in that it responds to TGF-h. About 6 hours after either RUNX3 (1-187) or antisense DNA, cellular proliferation was
SNU16 cells were treated with TGF-h, RUNX3 began to accumulate almost normal (Fig. 4C), suggesting that RUNX3 is required for
in the nucleus, whereas under the same conditions it remained in TGF-h-dependent growth inhibition. Other evidence indicates that
the cytoplasm in SNU5 cells, which are resistant to TGF-h stim- the growth inhibition of TGF-h-treated SNU16 cells is due to the
ulation (Fig. 4A and C; ref. 22). SNU16 cell number was greatly induction of both a proapoptotic gene6 and a cyclin-dependent
reduced after treatment with TGF-h compared with untreated kinase inhibitor (24).
SNU16 cells (Fig. 4C). To determine whether this phenomenon
is mediated by RUNX3, SNU16 cells were transfected with either
6
COOH-terminally truncated RUNX3, RUNX3 (1-187), or an T. Yano et al., submitted for publication.
Cancer Res 2005; 65: (17). September 1, 2005 7746 www.aacrjournals.org
Cytoplasmic Retention of RUNX3
Table 1. Subcellular localization of RUNX13 in gastric Discussion
cancer tissues We showed the expression patterns of RUNX3 protein in
human gastric epithelial cells and cancer cells by immunohis-
Negative, Positive in Positive in tochemistry with newly isolated monoclonal antibodies. Consis-
n (%) nucleus, n (%) cytoplasm, n (%) tent with our previous report of Runx3 RNA expression in the
adult mouse stomach (4), the RUNX3 protein is expressed most
Intestinal (n = 67) 24 (36) 12 (18) 31 (46) strongly in chief cells and surface epithelial cells and to a lesser
Diffuse (n = 30) 19 (63) 5 (17) 6 (20) degree in parietal cells in the normal human adult gastric
Total (n = 97) 43 (44) 17 (18) 37 (38)
mucosa. In this study, however, we observed a novel cytoplasmic
localization of a substantial fraction of RUNX3 in chief cells. In
line with our previous report, RUNX3 was not detected in 44% of
Next, the inhibitory effect of exogenous RUNX3 (1-187) on the the 97 gastric cancer cases tested. Surprisingly however, we
transcriptional activity of endogenous RUNX3 was monitored in a could only detect nuclear localization of RUNX3 in 18% of the
reporter assay using the TGF-h response element in the Ig Ca remaining cases. In the other 38%, it was detected primarily, if
promoter (Fig. 4D). This promoter has binding sites for RUNX and not exclusively, in the cytoplasm as an apparently nonfunctional
Smad proteins, which are indispensable for full activation of the Ig form.
Ca gene (5). Stimulation by TGF-h increased luciferase activity in a In many signaling pathways, signal transducers are transcrip-
manner dependent on both RUNX and Smad binding sites. As tion factors that are restricted by the nuclear envelope from
expected, reporter activities were inhibited in cells that expressed gaining access to target genes. Therefore, the nuclear import of
exogenous RUNX3 (1-187; Fig. 4D). These results suggest that RUNX3 transcription factors is an essential element of many of these
expressed in SNU16 cells is competent for transcription when
translocated into the nucleus. The fact that all the gastric cancer cell
lines studied do not have any mutations in the coding regions of
RUNX3 and that RUNX3 expressed in the cells is a full-size protein
(Fig. 3A) suggests that the cytoplasmic RUNX3 observed in gastric
cancer cells and cell lines is likely to be potentially functional. We
attempted to restore TGF-h responsiveness by reinstating RUNX3
in RUNX3-negative cell lines, but we were unsuccessful.
Unexpectedly, transfection of Flag-tagged RUNX3 (1-187) into
SNU16 cells inhibited the TGF-h-activated nuclear translocation of
endogenous RUNX3 (Fig. 4A). The R3-6E9 antibody detected only
endogenous full-length RUNX3 but not transfected RUNX3 (1-187),
which lacks the epitope recognized by R3-6E9 (Fig. 1A ).
Surprisingly, Flag-tagged RUNX3 (1-187 amino acids) also was
not translocated into the nucleus (Fig. 4A; see Discussion for
possible explanation).
Cytoplasmic retention of RUNX3 increases the tumorige-
nicity of SNU16 cells. We examined the effect of the RUNX3
(1-187)–mediated cytoplasmic retention of endogenous RUNX3
on the tumorigenicity of SNU16 cells by using xenografts in nude
mice. As shown in Fig. 5A and B, SNU16 cells stably expressing
RUNX3 (1-187) formed significantly larger tumors than those
formed by control SNU16 cells (P < 0.05). Control SNU16 cells
formed small-sized diffuse tumors, in which the accumulation of
endogenous RUNX3 in the nucleus was observed by immuno-
detection with R3-6E9 antibody (Fig. 5C). In contrast, endoge-
nous RUNX3 was detected mainly in the cytoplasm of larger
tumors formed by SNU16 cells expressing RUNX3 (1-187; Fig.
5C). Moreover, some nuclei in the tumors formed by control
SNU16 cells showed apoptosis as revealed by the TUNEL method
(data not shown). Endogenous RUNX3 was very weakly
detectable in tumors formed by MKN74 cells (Fig. 5C) and
NUGC3 (data not shown). Because both of these cell lines
express Smad2, Smad3, Smad4 and the TGF-h type II receptor
(data not shown), their tumorigenicity is consistent with the lack
of RUNX3 expression.
Figure 3. Immunodetection of endogenous RUNX3 in gastric cancer cell
Taken together, these observations clearly show that the lines. A and B, Western blot analysis with the R3-5G4 antibody on whole cell
tumorigenicity of SNU16 cells is enhanced by the cytoplasmic extract of indicated cell lines (A); cytoplasmic (c ) and nuclear (n ) extracts
retention of RUNX3, suggesting that its tumor suppressor function of SNU5 and RF48 cells (B). C, immunofluorescence analysis with the R3-6E9
antibody of the MKN1, MKN45, SNU5, SNU16, NCI-N87, RF48, MKN28, and
can be attenuated by mislocalization of the protein, even in the SNU1 cells. Nuclei were visualized by staining with 4V,6-diamidino-2-
presence of wild-type RUNX3. phenylindole (DAPI).
www.aacrjournals.org 7747 Cancer Res 2005; 65: (17). September 1, 2005
Cancer Research
Figure 4. Nuclear translocation of RUNX3 in
SNU16 cells induced by TGF-h. A, distribution
of endogenous RUNX3 in control SNU16 cells,
SNU16 cells expressing RUNX3 (1-187 amino acids),
and SNU5 cells as revealed by immunofluorescence
using R3-6E9 and the distribution of exogenous
RUNX3 (1-187 amino acids) in SNU16 cells
expressing RUNX3 (1-187 amino acids) as revealed
by immunofluorescence using an anti-Flag antibody
over a period of 24 hours after treatment with TGF-h
(2 ng/mL). B,left, immunodetection of exogenous
RUNX3 (1-187 amino acids) by Western blot
analysis with the anti-Flag antibody; right,
endogenous RUNX3 expression is reduced in
SNU16 cells expressing antisense RUNX3 DNA as
detected by RT-PCR. C, sensitivity of control SNU16
cells, SNU16 cells expressing RUNX3 (1-187 amino
acids), SNU16 cells expressing antisense DNA of
RUNX3 , and SNU5 cells to TGF-h. The growth of
each cell type in the presence (solid columns ) of TGF-h
(2 ng/mL) for 48 hours is normalized to growth in
its absence (open columns). D, luciferase activities
of (ThRE-WT)3 (WT ), (ThRE-mP)3 (mR ), and
(ThRE-mS)3 (mS ) reporter constructs (see Materials
and Methods) in control SNU16 cells (open columns )
and SNU16 cells expressing RUNX3 (1-187 amino
acids; solid columns ) over a period of 24 hours
after treatment with TGF-h (2 ng/mL). All luciferase
activities were normalized to the activity of luciferase
expressed from the vector pRL-TK as an internal
transfection control.
pathways.Transcription factors localized in the cytoplasm are factor. In these cells, it is known that the components of the
thought to be in a basal, inactive state. For transcription factors, TGF-h signaling cascade are often altered. Reduced expression
such as signal tranducers and activators of transcription (STAT) and of TGF-h type I/II receptors in MKN1 and Smad4 in MKN45
Smads, which require receptor-mediated phosphorylation for and NCI-N87 cells and mutation of the TGF-h type II receptor
conversion to the active state, the cytoplasm constitutes a reservoir in SNU5 cells have been described (22, 26).7 RUNX3 was
for the storage of unstimulated forms (25). imported into the nucleus of SNU16 cells only several hours after
The observation that a substantial fraction of RUNX3 resides stimulation with TGF-h, suggesting that TGF-h effect may be
in the cytoplasm of chief cells suggests that these proteins are indirect and that there might be more proximal signals that
in a basal state. TGF-h was identified as an agent that stimulate RUNX3 for nuclear translocation. Nevertheless, it is
stimulates the nuclear translocation of RUNX3. The cytoplasmic remarkable that many of the components of the TGF-h signaling
retention of RUNX3 observed in many cases of gastric cancer cascade are frequently impaired in gastric cancer cells, which
may be due to one or more missing components in a signaling prompts the conclusion that this cascade is primarily involved
pathway required for RUNX3 nuclear localization or by the in regulating the nuclear translocation of RUNX3. It is important to
presence of factors that impede the function of these
components. In the gastric cancer–derived cell lines MKN1,
MKN45, SNU5, and NCI-N87, RUNX3 is expressed but localized
to the cytoplasm, presumably as a nonfunctional transcription 7
H. Ida, unpublished data.
Cancer Res 2005; 65: (17). September 1, 2005 7748 www.aacrjournals.org
Cytoplasmic Retention of RUNX3
determine why it takes longer for RUNX3 to be translocated into the How is RUNX3 held in the cytoplasm? Recently, accelerated
nucleus of SNU16 cells after TGF-h stimulation. Because SNU16 is a osteogenesis has been observed in STAT1-null mice. It was found
gastric cancer–derived cell line, there may be a mild defect in this that, in its latent form, osteogenic Runx2 is bound to STAT1, which
signaling cascade. retains Runx2 in the cytoplasm. In the absence of STAT1, Runx2 is
Because the TGF-h–induced signal could be transmitted to translocated into the nucleus, where it stimulates osteogenesis (27).
targets in SNU16 cells, we examined the significance of the These results clearly show that the cytoplasmic retention of Runx2
cytoplasmic retention of RUNX3 by comparing SNU16 cells stably by STAT1 attenuates Runx2 function. A further involvement of
expressing RUNX3 (1-187) with parental cells in a nude mouse STAT signaling in gastric carcinogenesis has also been described in
assay and found that cells expressing RUNX3 (1-187) induced a recent study using gp130 mutant mice (28). It is attractive to
larger tumors than did control cells. We confirmed that RUNX3 speculate that one of the STAT proteins interacts with RUNX3 and
was primarily in the cytoplasm of cells from RUNX3 (1-187)– modulates its function in gastric epithelial cells.
induced tumors, whereas a substantial fraction of RUNX3 was in How does exogenous RUNX3 (1-187) inhibit the nuclear
the nucleus of cells from control tumors. These results are translocation of endogenous RUNX3 in SNU16 cells? We presume
consistent with the notion that RUNX3 does not elicit tumor that the agent that retains RUNX3 in the cytoplasm interacts
suppressive activity when it is restricted to the cytoplasm, with the NH2-terminal 187–amino acid region of the protein.
suggesting that the cytoplasmic retention of RUNX3 that we One possibility is that RUNX3 must be modified, for example, by
observed in a large fraction of gastric cancer cases is a novel phosphorylation, within the missing COOH-terminal region to
mechanism for inactivating RUNX3 function. allow its translocation into the nucleus. It is also worth noting
that RUNX3 interacts with R-Smads via its COOH-terminal
region (5), suggesting that COOH-terminally truncated RUNX3 is
retained in the cytoplasm because it can no longer interact with
Smads. Transcriptionally active members of the RUNX family of
proteins are heterodimers composed of a and h subunits. The h
subunit, PEBP2h/CBFh, protects the RUNX proteins from
proteolysis (23, 29). Furthermore, COOH-terminally truncated
RUNX proteins have a higher affinity for PEBP2h/CBFh than for
full-length RUNX proteins (23). Therefore, the sequestration of
PEBP2h/CBFh by an excess of exogenous RUNX3 (1-187) may
inhibit the nuclear translocation of endogenous RUNX3. Further
studies are required to clarify the exact mechanism.
The cytoplasmic retention of RUNX3 is a useful ‘‘molecular
marker’’ to detect inactivity of RUNX3 as a tumor suppressor and
impairment of the TGF-h signaling pathway. Therefore, the
monoclonal antibodies R3-6E9 and R3-2A4 are powerful tools for
the characterization of gastric cancer cells. We are currently
studying whether these antibodies can be used to detect early stages
of gastric cancer and precancerous states in gastric epithelium.
Recently, the possible involvement of RUNX3 in other types of
cancer as well as in gastric cancer has been reported. These
include lung (30–32), breast (32), pancreas (33), liver (32, 34),
bile duct (33), colon (35, 36), gallbladder (37), prostate (38),
larynx (32), esophageal (39), and gastric (40, 41) cancers and
testicular yolk sac tumors in infants (42). These studies focused
on the lack of RUNX3 expression caused by hypermethylation of
the RUNX3 promoter region. In the light of the results presented
here, it would be interesting to study the subcellular localization
of RUNX3 in these cancers using R3-6E9 and R3-2A4 antibodies.
This might reveal that RUNX3 is much more widely inactivated in
cancer than previously thought, which would pose further
questions, and perhaps shed light on the true role of RUNX3 in
cancerogenesis.
Acknowledgments
Figure 5. The increase of tumorigenicity of SNU16 cells promoted by the Received 3/2/2005; revised 5/25/2005; accepted 6/16/2005.
cytoplasmic retention of RUNX3. A, tumors formed by control SNU16 cells Grant support: Agency for Science, Technology and Research, Singapore and
(white arrowheads ) and SNU16 cells stably expressing RUNX3 (1-187 amino Human Frontier Science Program RGP0375/2001-M202. Q. Liu, M. Salto-Tellez, and
acids; black arrowheads ) in nude mice. B, sizes of tumors formed by control K.G. Yeoh received funding support from SCS Grants GN-15 & MN-05, awarded by the
SNU16 cells and SNU16 cells stably expressing RUNX3 (1-187 amino acids) in Singapore Cancer Syndicate, Agency for Science, Technology and Research, Singapore.
nude mice. Columns, mean of seven determinations; bars, SD. C, detection The costs of publication of this article were defrayed in part by the payment of page
of endogenous RUNX3 in tumors formed by control SNU16 cells, SNU16 charges. This article must therefore be hereby marked advertisement in accordance
cells expressing exogenous RUNX3 (1-187 amino acids), and MKN74 cells in with 18 U.S.C. Section 1734 solely to indicate this fact.
nude mice by immunofluorescence with R3-6E9. Nuclei were visualized by We thank Dr. Y. Groner for the kind gift of murine Runx3 cDNA, and E. Tai, P.Y.
staining with DAPI. Chong, B.H. Neo, and Y.K. Loh for technical assistance.
www.aacrjournals.org 7749 Cancer Res 2005; 65: (17). September 1, 2005
Cancer Research
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